Process for differentiating the isoenzymes of lactic dehydrogenase



June 11, 1968 A. 1.. BABSON 3,388,044

PROCESS FOR DIFFERENTIATING THE ISO-ENZYMES OF LACTIC DEHYDROGENASE Filed Dec. 7, 1965 2 Sheets-Sheet 1 LA] 2 2 N .J A O A a 9-1 1'. O O u m 5 O 5 2% O O O 8 8 8 O o 02s 1v aouvaaosev INVENTOR ARTHUR L. BABSON ATTORNEY June 11, 1968 so 3,388,044

PROCESS FOR DIFFERENTIATING THE ISO-ENZYMES 0F LACTIC DEHYDROGENASE Filed Dec. 7, 1965 2 Sheets-Sheet 2 LL] E A D N o 1 g N E 3 4 D UJ a: O a D a 3% E .J O 2 r I. O

N Q Q 6 o o O o O O 3 0 029 1v aouvaaosav INVENTOR ARTHUR L. BABSON ATTORNEY 3,383,044 PRGCESS FUR DlFblEl-ZENTIATTNG THE 156- ENZYMEd @F LACTEQ BEHYDRGGENASE Arthur L. liabson, Morristown, NJsL, assigncr to Warner- Lamhert Pharmaceutical iCompany, Morris llains, Ni,

a corporation of Delaware Filed Dec. 7, 1965, Ser. No. 512,117 6 (Ilairns. (El. 195-1035) This invention relates to a novel diagnostic process and more particularly relates to a new and novel process for the rapid and accurate determination as well as the rapid and accurate diiferentiation of the tissue of origin of lactic dehydrogenase in body fiuids.

This invention also includes within its scope new and novel substrates useful in the above diagnostic process.

Lactic dehydrogenase, referred to hereinafter for convenience as LDl-l, is an enzyme found in most mammalian tissues. It catalyzes the conversion of L--lactic acid to pyruvic acid in the presence of nicotinamide adenine dinucleotide. The latter is known also as coenzyme I, cozymase and as diphosphopyridine nucleotide but for simplicity will be referred to hereinafter as NAD. In reduced form the latter is referred to for con venience as NADl-l The reaction by which Llactic acid is converted to pyruvic acid may be represented by the following equa tion:

L(+) lactic acid NADipyruvic acid NADH The enzyme LDH has been found to consist of five different subunits or iso-enzymes, the relative concentrations of which vary markedly with the tissue of origin. Each of these iso-enzymes may be identified by its electrophoretic mobility. Thus, LDH isolated from cardiac muscle, although catalyzing the same reaction, has a different iso-enzyme pattern from that isolated from skeletal muscle. While in a healthy human, these iso-enzymes are, for the most part, confined within the tissues, during abnormal growth of tissues where tumors are formed or in leukemia or other conditions such as myocardial infarction and hepatitis, for example, they are found to be present in the serum in appreciable concentration. It has been postulated that the passage of an enzyme into serum from an abnormal tissue of origin may be due to an excessive production of the enzyme by the abnormal tissues or to changes in the permeability of the abnormal cells resulting in enzyme leakage into extra-cellular fiuid and thence into general circulation. Accordingly, the determination of the level of serum lactic dehydrogenase provides valuable information to the clinician in the diagnosis of various diseases. Due to the difiiculty of pin-pointing with certainty the tissue of origin of the enzyme, much of the value in the differential diagnosis of diseases which cause elevations in serum LDH is lost.

According to my invention as described and claimed in copending application S.N. 417,439, filed Dec. 10, 1964, now US. Patent 3,326,777 a differentiation of the tissue of origin of serum LDH can be effected by measuring the activity of the iso-enzymes comprising LDH at different concentrations of lactic acid in the substrate. For example, LDH of cardiac muscle tissue origin is at maximum activity in a substrate of about 0.01 to 0.2 molar L(+)lactate concentration whereas the lactic dehydrogenase of liver tissue origin is most active in a substrate of 0.05 to 0.5 molar L(+)lactate concentration.

These ranges of activity are shown in accompanying FIGURE 1 which also indicates from the activity curves shown therein the concentration of maximum activity. These curves are obtained by plotting optical absorbance at 530 mu against a substrate containing 0.01 to 1.0 molar L(+) lactate. The samples utilized for determining these States Patent "ice curves are obtained by extracting human heart, liver or skeletal muscle at autopsy with an aqueous solvent. The resulting extracts are then added to pooled human serum and the resulting optical absorbance provided with various concentrations of the lactate substrate.

The expression molar as used herein and in the claims refers to a substrate solution which contains one mol of the substance in one liter of solution. Thus, 1 molar urea solution (1 M urea) contains 60.1 grams urea per liter and one molar L(+) lactic acid solution contains 90.1 grams of L(+) lactic acid per liter. The substrates used in the practice of this invention are prepared by dissolving the selected substance or substances in Water to give the desired final concentration.

While these curves are still valid, it has now been found that the ability to differentiate and distinguish between the iso-enzymes pattern of LDH from different sources by assaying the body fluid at high and low concentrations of lactate is remarkcc'ly enhanced by the incorporation in the reaction medium of from about 0.5 molar to about 2 molar of urea, and more especially in the region of low lactate concentration as described below.

The presence of urea at a lactate concentration of below 0.1 molar appears to have the effect of inhibiting the activity of LDH enzyme of liver origin as clearly shown in FIGURE 2 by the lower optical absorbance observed which is the index of enzyme activity in the test procedure described. The effect of urea in producing this inhibition and resulting differentiation is dramatically brought out when the activity curves of the same liver LDH enzyme assayed with or without urea are plotted and compared. This is clearly shown by comparing the activity curves of FIGURE 1 with those of FIGURE 2.

Thus, in FIGURE 1 the activity of liver LDH enzyme at various lactate concentrations is illustrated and the data is obtained by assaying the activity in various lactate concentrations from 0.01 to 1.0 molar in the absence of urea. At a lactate concentration of 0.015 molar, for example, the optical absorbance is about 0.685. As shown in FIGURE 2, at the same lactate concentration but in the presence of urea, an optical absorbance of about 0.140 is observed.

On the other hand, the curves plotted in FIGURES 1 and 2 show that heart LDH enzyme activity is not appreciably affected by the presence of urea. Because of the effect of urea on liver LDH enzyme in low substrate concentrations the differentiation in activity between these enzymes is quite marked and therefore easily recognized.

By incorporating urea into the substrate sharp but well spread end points are obtained with resulting advantages.

The practice of this invention is illustrated in the following detailed description.

In a typical situation where such a differential determination is to be effected in accordance with the method of this invention 0.050.2 ml. of patients serum or other body fluid such as spinal fluid is incubated at 37 C. with an aqueous substrate containing about 0.05 molar L(+) lactate, a bufier such as tris-hydroxymethylaminoethane to maintain the pH of the substrate at about 8 to 9, a small amount of nicotinamide adenine dinucleotide, a suitable chromogenic electron acceptor which acts as an indicator such as iodonitrotetrazolium chloride (INT) or nitro-blue tetrazolium and an intermediate electron carrier such as phenazine methosulfate. The chromogenic electron acceptor indicator exists in colorless form in its oxidized state and becomes colored when in a reduced state. For instance, INT becomes red when it is reduced. The resulting mixture is then incubated for 5 minutes at 37 C. after which the reaction is stopped by the addition of an acid such as, for example, 0.1 N aqueous hydrochloric acid. The optical absorbance is then determined at a wave length of 530 m in a suitable colorimeter. The intensity of the color produced is proportional to the LDH present in the body fluid being tested. The optical absorbance thus obtained is then compared with that produced by a control sample.

The control is prepared by incubating a control sample of the body fluid with potassium oxalate with or another suitable oxalate in place of the lactate and with disodium ethylene diamine tetraacetic acid (EDTA). The oxalate ion inhibits any lactic dehydrogenase activity and the EDTA prevents the precipitation of any calcium oxalate formed thereby providing a blank with all the ingredients except enzyme activity for optical comparison. It the comparison of the optical absorbance of the control sample indicates the presence of elevated lactic dehydrogenase, this assay procedure is repeated in an analogous fashion but in parallel employing a divided sample in which one substrate contains a high concentration of lactate and the other a lower concentration of lactate and urea. Thus, for example, a solution containing 0.6 molar L(+) lactate may be employed as the substrate for one determination while a solution containing 0.02 molar L(+) lactate and 1 molar urea may be employed as the substrate for the other. The assay procedures are then carried out as described, and the optical absorbances are determined. Since the enzyme will have different substrate optima activities depending on its origin, the origin may be readily determined by comparing the optical absorbances produced.

Thus, for example, referring to FIGURE 1 it will be seen that liver LDH when assayed in a substrate containing 0.6 molar L(+) lactate, an optical absorbance of about 0.600 is obtained. The same liver enzyme when assayed in a substrate containing 0.02 molar L( lactate and 1 molar urea, as brought out in FIGURE 2, yields an optical absorbance of about 0.140. The ratio of these two optical absorbances is about 4.4:1.

On the other hand heart LDH when assayed in a substrate containing 0.6 molar L(+) lactate, an optical absorbance of about 0.360 is obtained, and when assayed in a substrate containing 0.02 molar L(+) lactate and 1 molar urea, an optical absorbance of 0.720 is obtained.

The ratios of these two optical absorbances is about 0.5 :1. In other words, when a ratio significantly above about one is obtained, it indicates liver involvement and a ratio significantly less than about one is obtained, it indicates heart involvements. Thus a differential diagnosis of the tissue of origin of LDH enzyme is readily obtained.

It is frequently advantageous also to include a surface active agent in the substrate to stabilize the color formed. Surface active agents such as polysorbate 80, albumin or ethoxylated fatty alcohols from 0.005 to 0.5% by weight may be added.

In the above-described process, the L(+) lactate employed may be, for example, sodium lactate, potassium lactate, calcium lactate and the like. If the substrate is a racemic mixture of dl-lactate, twice the molar concentration is employed. Thus, when 0.1 molar is used for the L-form, 0.2 molar is used for the racemic (d1) mixture.

In order to further illustrate the practice of this invention, the following example is given:

Example The various reagents employed in the determination of tissue of origin of lactic dehydrogenase are prepared as follows:

(1) The color reagent is prepared by dissolving 50 mg. of INT in about ml. of water with constant stirring if necessary to facilitate dissolution. To this solution 125 mg. of NAD and 12.5 mg. of phenazine methosulfate are added. The resulting solution is made up to a final volume of ml. with more water in a volumetric flask. Since this reagent is sensitive to light it 4 is protected from light at all times to avoid decomposition.

(2) The buffer reagent is prepared by dissolving 12.1 g. of tris-hydroxymethylaminomethane in water. The pH of this solution is then adjusted to 8.2 with 3 N HCl and diluted to a final volume of 100 ml. with more water.

(3) The high LDH substrate is prepared by dissolving 18 g. of crystalline L(+) lactic acid in about 40 ml. water. This is adjusted to a pH 5.5 with 5 N sodium hydroxide and diluted to 100 ml. with water. The substrate is stored under refrigeration.

(4) The low LDH substrate is prepared by taking 1 ml. of reagent #3 and 12 g. urea diluting to 100 ml. with water.

(5) The control reagent is prepared by dissolving 0.2 g. potassium oxalate and 0.2 g. ethylene diamine tetraacetic acid disodium dihydrate in 100 ml. of water.

PROCEDURE FOR TESTING 0.1. ml. of the patients serum or other body fluid is pipetted into each of three test tubes containing 0.2 ml. of the buffer reagent. 0.5 ml. of each substrate is added to two tubes and 0.5 ml. of the control reagent to the third. After thoroughly mixing each of these two tubes, they are incubated at 37 C. for 8 to 10 minutes. At precisely timed intervals 0.2 ml. of the color reagent is added and they are allowed to be incubated together for an additional 5 minutes at 37 C. After the 5 minutes incubation period 5 ml. of 0.1 H01 acid is added with thorough mixing to stop the enzyme reaction. The optical absorbance of the control and the two substrate tubes is determined at a wave length of about 500-540 m The ratio of the optical absorbance obtained from high LDH substrate and low LDH substrate is then calculated. A ratio of significantly above one suggests liver LDH enzyme Whereas a ratio of significantly less than one suggests heart LDH enzyme.

It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of my invention.

Having described my invention, what I desire to secure by Letters Patent is:

1. Process for differentiating the tissue of origin of lactic dehydrogenase in a body fluid which comprises the steps of measuring the activity of said enzyme in a substrate containing about 0.5 molar to about 2 molar L(+) lactate and comparing the activity thus produced with that produced by said enzyme in a substrate containing about 0.05 molar to about 0.002 molar of L(+) lactate and about 0.5 molar to about 2 molar urea.

2. Process for differentiating the tissue of origin of lactic dehydrogenase in a body fluid which comprises the steps of measuring the activity of said enzyme in a reaction mixture containing about 1 molar of L(+) lactate and comparing the activity thus produced with that produced by said enzyme in a substrate containing about 0.01 molar of L(+) lactate and 1 molar urea.

3. Process for differentiating the tissue of origin of lactic dehydrogenase in a body fluid which comprises the steps of measuring the activity of said enzyme in a substrate having a pH of 8 to 9 containing about 1 molar of L(+) lactate in combination with small amounts of nicotinamide adenine dinucleotide, a chromogenic electron acceptor, an intermediate electron carrier and a surface active agent and comparing the optical density thus produced with that produced by said enzyme in a substrate having a pH of 8 to 9 containing about 0.01 molar of L(+) lactate and about 1 molar urea in-combination with small amounts of nicotinamide adenine dinucleotide, a chromogenic electron acceptor, an intermediate electron carrier and a surface active agent.

4. Process in accordance with claim 3 wherein said surface active agent is a member of the group consisting of egg albumin, polysorbate and ethoxylated alcohol.

3,388,044 5 6 5. Process in accordance with claim 3 wherein said References Cited electron carrier is Phenazine methosulfate' Wilkinson: An Introduction to Diagnostic Enzy- 6. Process in accordance with claim 3 wherein said 1 154457 1962 m0 gy, PP' chromogenlc electron acceptor 1s a member of the group consisting of iodonitrotetrazolium chloride and nitro- 5 ALVINE' TANENHOLTZ, Primary Examiner blue tetrazolium. 

1. PROCESS FOR DIFFERENTIATING THE TISSUE OF ORIGIN OF LACTIC DEHYDROGENASE IN A BODY FLUID WHICH COMPRISES THE STEPS OF MEASURING THE ACTIVITY OF SAID ENZYME IN A SUBSTRATE CONTAINING ABOUT 0.5 MOLAR TO ABOUT 2 MOLAR L(+) LACTATE AND COMPARING THE ACTIVITY THUS PRODUCED WITH THAT PRODUCED BY SAID ENZYME IN A SUBSTRATE CONTAINING ABOUT 0.05 TO ABOUT 0.002 MOLAR OF L(+) LACTATE AND ABOUT 0.5 MOLAR TO ABOUT 2 MOLAR UREA. 